The best techniques for identifying sites of contact in protein complexes are x-ray diffraction and nuclear magnetic resonance. However, those techniques may not be available for large complexes, due to limited sample amount, aggregation, insolubility, posttranslational modification heterogeneity, lack of suitable crystals, etc. Most alternative methods are based on exposing the complex to some sort of chemical perturbation (e.g., chemical reactivity toward general or specific agents, cross-linking, and the like). The contact surfaces may then be located as those sites that are linked or become protected against chemical reactivity on formation of the protein complex. The most generally applicable measure of solvent exposure is exchange of backbone amide hydrogens for deuteriums, because it is least dependent on the amino acid sequence. Major current difficulties for the H/D exchange method include: back-exchange of H for D during separation of proteolytic fragments for analysis;mass resolving power too low to separate and identify partially deuterated peptides by mass spectrometry;incomplete sequence coverage by pepsin cleavage (pepsin is usually used due to its activity at low pH after H/D exchange has been quenched);and the difficulty and duration of acquiring and interpreting the data. In this project, we shall combine several improvements: supercritical fluid chromatography to eliminate back-exchange;isotopic depletion and ultrahigh mass resolving power to simplify identification of peptides, a suite of enzymes of different proteolytic specificity to better span the sequence;and automation of data collection and data analysis. Relevance. It is becoming increasingly evident that protein complexes and assemblies play a key role in many human diseases: e.g., the protein "capsid" that protects RNA in the AIDS virus and in the biological "motors" that transport essential components into a cell or virus. A first step in understanding (and eventually controlling) those functions is to identify the sites of contact that hold proteins together. This project presents several new approaches for such "mapping", along with some suggested initial applications that could point to future targets with pharmaceutical applications. This project is multidisiplinary and pulls together several distinguished collaborators with focused research projects that will benefit directly from H/D exchange with high-resolution mass analysis.